Method of producing butanol and acetone by fermentation



Patented Aug. 11, 1931 UNITED STATES PATENT oFF1cE STEFAN BAKONYI, F DESSAU, GERMANY 'ME'IHOD OF IR ODUCING BUTAN'OL AND ACETONE BY FEBMENTATION No Drawing. Application filed December 21, 1928, Serial No. 327,751, and in Germany October 22, 1928.

For the purposes of producing butanol and acetone from saccharine or amylaceous raw materials by fermentation there has already been proposed and used a great number 5 of bacteria, for instance, Bacillus vbutg licus Fitz (German Patent No. 323,533), Bacillus Weizmann (British Patent No. 4845/1915), bacillus butylz'cus Boinot-Firmin (U. S. Pate'nt No. 1,385,888), Bacillus acel'o-butg lz'cum (U. S. Patent No. 1,427,595), Bacillus bu ty group will be set forth in the following specification. c

The bacteria of the butylobacter group form vegetative cells, 2-3 microns in length and ,4 micron in diameter. The bacteria lying in pairs form an obtuse angle, resems bling to M yambacterz'um tuberculosis. Contrary to all other above named species these cells form under ordinary conditlons neither chains nor filaments. An important feature is the gramcoloring: if, for instance, a corn mash of 5% is inoculated with spores, the young cells are at first 'grampositive (blue). After 24 hours however they get a red-violet 40 color and become red more and'more every hour, they are sharply red (gramnegative) 42 hours later. This color is retained during 1-3 hours, then they assume again the redviolet color. Finally, after 48 hours, they become anew grampositive (blue)v The formation of spores takes place after 24 hours. The spores lie eccentrically and form a cylindric figure about 1 micron in length and micron in diameter.

The fermenting power in concentrated 5o mashs is rather characteristic. The species of bacteria hitherto described are enabled to ferment a 5 to 8% mash with a yield of A, of the starch employed and this gives a yield increased by 40-43% at a doubled 56 concentration. This double concentration saves in the production of butanol and acetone by fermentation with butylobacter a good half of steam and power consumption. Considering the greatly increased yield the use of butylobacter is twofold economical.

An other important feature of the butylobacter group is the ability of its members to accomodate themselves to the actual raw material to be fermented. For instance, a species grown on maize (butylobacter zeae Bakonyi) forms a plenty of spores after having been transferred4 or 5 times to concentrated potato soup in intervals of 24 hours each time. The fermenting power then is as good as in corn mashs. But also in raw materials, which can be fermented only with difliculty, as for instance in a diluted cane sugar molasses (blackstrap) they show without nutrient additions after being 4 times transferred in intervals of 24 hours each time a strong fermentation and copious formation of spores.

The preparation of the butylobacter may be carried out somewhat according to the following method, whereto the usual prim ciples of bacteriological technical science ture are obtaine of -1-2% of butanol prevents the growth ofall microorganisms, which are less resistant to butanol. The preparation of culture may be carried o ut'by selecting a natural mixed culture and attaching the same to a solid substrata which is subject to change by the action of the.'biochemical agents such as particles of grain. The substrata should be freely movable in asuitable solution. A culture thus is formed. Effective inciters may be obtained by systematic selection, viz by first adding to the culture to be employed and the nutrient substrata small quantities of the butanol and acetone to be produced. Automatic selection thus takes place. The bacteria which are non-resistant to' butanol and acetone are eliminated while the resistant bacteria survive. After suitable development of the bacteria a further selection under the microscope is desirable. By preparation of a number of separate samples and suitable selection of the best of those samples the strongly degenerated (granulated or deformed) individuals are eliminated and the microorganisms which show uniform protoplasm and normal shape are chosen for further development. A further microscopic investi ation of the samples using fixed and colored preparations is desirable. The samples to be finally employed are those in which the microorganisms show good forma tion of culture and fixation to the solid nutrient substrata. A sterilized nutrient substrate to which about 1% of butanol and acetone is-added is inoculated with these selected samples. The inoculated samples are incubated for twenty-four hours and the microscopio selection is preferably repeated to further improve the qualityof the bacteria. 7 Operation of microsco ic selection and m-.

cubation of the selecte cultures is repeated until cultures of satisfactorily normal appearance showin a strong formation of cul- The same bacteria as repeatedly mentioned above cannot be repared according to the method describe by the spores of the butylobacter are very s'ensitive to heat, much more sensible'in any case than the spores of other hitherto known species. Heating up to 100 C. as prescribed by Weizmann produces a rather unfavorable effect: generally the spores are killed and if they survive they show a retarded and'decreased fermenting effect. Moreover it is necessary'to select a carrier of bacteria fit for these purposes. Hitherto I have found four species of the butylobacter:

1. butylobacter zeae Bakonyi, prepared on Hungarian maize 1924:,

2. butylobacter betae Bakonyi, prepared on beets (Beta vulgaris) Kreis Zerbst 1928,

3. butylobacter sinense Bakonyi, prepared on J afi'a oranges 1928,

eizmann (British. Patent 4845/1915) for 4. butylobacter 'solani Bakonyi, prepared on German potatoes, Kreis Zerbst 1928. But I have not succeeded in winning the butylobacter from German maize, because it can be found, as I presume," only on such fruits, as are gathered on a good humus soil or are in continual touch with the soil themselves (beets and potatoes).

The best method of preparation is the following:

On the bottom of a sterilized test tube of about 40 cm. in length, an aseptically taken sample from the (unsterile) raw material is transferred and covered with 20 cm. in height of a 5% sterile corn mesh containing about 12% of butanol, whereupon the 813011313661 test tubes are to be incubated-at 37 C. for

about 24 hours. In the test tubes an active fermentation sets in if butylobact'er or other inciters of fermentation processes are present. The mlcroscoplcal figures are with regard to the addition of butanol rather simple:

1. clumpy-short bacteria, different strepto-l different kinds according to the well known agar plates method, otherwise the culture is-transferred for 24: hours to a fresh butanol containing mash all those being removed which are less resistant against butanol.

Upon plates butylobacter grows only anaerobically and can be separated without difficulty by the following morphological characteristics from the other species.

1. The vegetative cells are considerably shorter and slenderer than all the other hitherto known butanol acetone bacteria. The bacteria lying in pairs form an obtuse angle, resembling to Mymobactem'm tuberculosis. They do not form chains and show the said rather characteristic gramcoloring (positive, then negative, then again positive).

2. The spores belong likewise to the smallest ever formed by butanol-acetone bacteria,

they have the form of a cylinder, whilst the spores of the other kinds are oviform. Only the Bacillus butylz'cus Boinot-Firmin has spores of about the same size and form; however the butylobacter forms only one spore, Bacillus butg/Zimts B. F. frequently two, butylobacter has rounded, B. F. unrounded cells, butylobacter forms neither chains nor filaments as B. F. and finally butylobacter is nearly by half shorter and slenderer than B. F. In the following table the characteristics of the best known species are assembled in order to facilitate the identification of the butylobacter.

' Oioshidinm r Bacillus Weiz- Bacillm butyl. Bacillus buty- Baa. acetobutyiiman "13. F. m Butyloimetaz- Author Weizmann Boi m1 5 Hort on. "rm-slam... Bakonyi. Patent Bri t rat. 4845/ U. 8. Pat.1,385, U 3 Pat.1,537,- U g Pat. 1,427,- 11. Pat.1,655,

1 1 Cells 3-3) 3-5- 3-4- 2-4-1-2 3-10- 2-3- (micron)- 0, 3-0, 6 0, 0, 4-0, B 0, 76- 0, 25-0, 3 Spores (micron) 2.2-1, 2 1-1, 2-0, 2.2- 1,2 1, 6-1, 2 2-2, 5-1 1-0,5-'0,7 Figure of Rounded Unrounded Rounded Rounded- Rounded Rounded. Formation of chains Exist--- xist xist Exist--. Finn None. Figure of spores Ov l Cylindr Ov l Ov l Ov l Cylindr Quantity of apores 1 1-2 1 1 1 Gramooloring. P03 1 Pos.- Poe" Pm Poe. then nega- Poe. then neg.. tive. then again pol.

' The method of producing butanol and aceteria of, the 'butylobacter group and fe rtone by fermentation may be carried .out as mentmg the carbohydrate mash with said follows: .0,000'kgs. corn meal (containing 6000 kg. starch) suspended in the threefold quantity of water are boiled for one to two hours under two atmospheres pressure, blown out in a fermentation vatand diluted up to about 80,000 liters. By cooling down the temperature is reduced to about 37 C. and the wort is now inoculated with a laboratory culture of with certain quantity of fermenting wort. In the latter case the fermentation 'takes place at once and is completed after 30-40 hours. The formed products (butanol and acetone) are separated by distillation. The yield is 2500-2700 kgs.

In the same manner all the other cereals, potatoes, molasses etc. may be used.

When using molasses as a raw material for the fermenting process a previous heating of the molasses soup for about half an hour up to 100 C. will permit a vigorous and satisfactory fermentation.

I claim:

1. The method of preparing bacteria of the butylobacter group which comprises covering an asceptically taken sample from raw material containing bacteria with 5% sterile corn mash containing from 1 to 2% of butanol, incubating the same at a temperature of about 37 C. for about twenty-four hours and separating the bacteria of the butylobacter group by the agar plates method.

2. The method of preparing bacteria of the butylobacter group which comprises subject ing raw material containing mixed. bacteria to a sterile mash containing. approximately 1 to 2% of butanol and incubating at a temperature approximately 37 C. c I

3. The method of producing butanol .and acetone which comprises sterilizing and coolin to a suitable temperature a suitable carboiydrate, inoculating the same with inciters of fermentation consisting the butylobacter, group pro need and separated by covering an asceptically taken sampie from raw material carrying bacteria with 5% sterile corn mash contaimng from 1 to,

2% of butanol, incubating the same at a temperature ap roximating 37 C. for about twenty-four ours and separating the bacof bacteria of.

bacteria. 4 r

4. The method of producing butanol and acetone which comprises boiling a starchy material in water to prepare it or fermentation, cooling said starchy material and inoculating and fermenting the same with bacteria of the but lobacter group produced and separated y covering an asceptically taken sample from raw material containing bacteria with 5% sterile-cornmash containing from 1 to 2% of butanol, 1ncubating the same at a temperature approximating rating the bacteria of the butylobacter group.

6. The method of producing butanol and acetone by fermentatiom of carboh drates which comprises cookin the carbohy rate to gelatinize the same, coo ing and inoculating the cooled carbohydrate with a mixture of bacteria'of the butylobhcter group and fermenting the carbohydrate. I

b7. The method of preparing bacteria of .t e

re arin bacteria of the butylobacter p p g group which comprises subteria to a sterile mash containing approxi- Jmately 1 to 2% of acetone and incubating at a temperature approximately 37 C. p

the bacteria of the butyljectlng raw material containing'mixed bacbutylobacter group which comprises covering an asceptically taken sample from 9. The method of producing. butanol and acetone 'by fermentation of carbohydrates which comprises-sterilizinglthe carbohydrate, inoculating the same 'wit bacteria of the I butylobacter group prepared from raw material containing mixed bacteria by subjecting. the same to a sterile mash containing small quantities of butanol and acetone and in cubating the same at a suitable temperature, 10 and fermenting the carbohydrate.

10. The method of producing butanol and acetone by fermentation of carbohydrates which comprises sterilizing the carbohydrate, inoculating the same with bacteria of the 15 butylobacter group .pre ared from rawmate rial containing mlxed acteria by subiectinfi the same to a sterile mash containing sma 1 quantities of butanol and incubating the same at a suitable temperature, and ferment:

1 ing the carbohydrate.

11. The met od of producing butanol' and acetone by fermentation of carbohydrates which comprises sterilizing the carbohydrate," inoculating the same with-bacteria of the butylobacter group prepared from-raw material containing mixed bacteria by subjecting I the same to a sterile mash containing sina quantities of acetone and incubating thesarne at a suitable temperature, and fermenting the 30 carboh drate- I 12. he. method ofproducing butanol and acetone by fermentation-of a suitable car.- bohydratewhich comprises developinga culture of bacteria-of the butylobacter group by .85 subjecting 'raw1 .material containing mixed bacteria, to 'aznutritive mash containing -a small quantity of butanol. incubating the same at a suitable temperature, selecting the best developed bacteria and further incubating the selected bacteria in a nutritive mash containing a small quantity of, butanol to develop a sub-culture and fermenting the carbohydrate with a subculture of the bacteria so produced.

e5 13. The method of producing butanol and acetone by fermentation of a suitable carbohydrate which comprises developing a culs ture of bacteria of the butylobacter group by subjecting raw material containing mixed bacteria-.to a nutritive mash containing a small quantity of acetone. incubating the same at a suitable temperature, selectmg-the developed bacter a and further incubating the selected bacteria in a nutritive mash con 53 taining a small quantity of acetone to develop a sub-culture and fermenting the carbohy- I drate with a sub-culture of the bacteria so Produced.

In testimony whereof I afix my signature. STEFAN BAKONYI. 

